This invention relates to compounds, processes for preparing them and their use as enzyme substrates.
The majority of metabolism based drug interactions are a result of inhibition of cytochrome P450 enzymes. Drug interactions involving individual P450 enzymes can be predicted using in vitro methods. Typical in vitro P450 enzyme assays involve incubation of an appropriate substrate with a source of enzyme. Traditionally time consuming chromatographic methods have been used for metabolite detection in these incubations. More recently the availability of fluorimetric plate readers has facilitated tne higher throughput of enzyme assays in general. Adapting P450 assay to fluorescent plate reader technology requires the identification of substrates with appropriate fluorescent products for individual enzymes. Among the xenobiotic-metabolising cytochromes P450, CYP2C9 is one of those commonly responsible for the metabolism of drugs.
3-Cyano-7-ethoxycoumarin has been described for high throughput CYP2C9 inhibition screening (Crespi et al. Anal. Biochem., 1997: 248, 188-190). However, the rate of 3-cyano-7-ethoxycoumarin metabolism by CYP2C9 is low and the extent of 3-cyano-7-ethoxycoumarin O-dealkylase inhibition does not always correlate well with a solid-phase extraction assay for CYP2C9, thus 3-cyano-7-ethoxycoumarin is not suitable for high throughput screening.
Certain compounds have now been identified which are improved substrates for CYP2C9 and which are of use for configuring high throughput inhibition screening assays.
According to the present invention there is provided an assay for testing for inhibitors of the enzyme CYP2C9 which comprises contacting the enzyme and a compound of formula (I): 
wherein R1 represents C1-2alkyl, with a test compound and measuring inhibition of O-dealkylation of the compound of formula (I) by the enzyme.
In a preferred aspect of the invention R1 is methyl.
Generally, the rate of O-dealkylation of the compound of formula (I) in the absence of test compound will be known, as will the extent of O-dealkylation at given time points. The assay may test for inhibition of O-dealkylation continuously or at specified time points.
O-Dealkylation of the compound of formula (I) following incubation with CYP2C9 gives a readily quantifiable fluorescent product of formula (II): 
which can be scanned with suitable excitation and emission wavelengths, for example an excitation wavelength of 410 nm and an emission wavelength of 510 nm.
The assay may be carried out either in solution or utilising a solid support. When the assay is carried out in solution suitable solvents include methanol, acetonitrile and DMSO.
The test compound may be pre-incubated with enzyme prior to the addition of the substrate, or alternatively the substrate may be added simultaneously. Final concentrations of enzyme and substrate are calculated so as to achieve a suitable rate of processing for carrying out the assay. If desired, the reaction may be stopped, for example by addition of acid or solvent. The fluorescent product of formula (II) may be analysed using any conventional system of fluorescence detection, for example a multi-well plate/fluorescent plate reader.
The compounds of formula (I) and (II) are novel and as such also form part of the invention.
The compounds of formula (I) and (II) may be prepared by conventional methods, for example as shown in Scheme 1: 
Thus according to a further aspect of the invention there is provided a process for the production of a compound of formula (I) or (II) which comprises:
a) reaction of resorcinol and a dialkyl trifluoroacetosuccinate wherein the alkyl groups are independently selected from C1-2 alkyl, in the presence of polphosphoric acid;
b) for compounds of formula (I) reaction of the resulting 7-hydroxycoumarin with a compound of formula R1Hal, wherein R1 is C1-2 alkyl and Hal is halogen e.g. iodine or bromine; and
c) ester hydrolysis to give the acid of formula (I) or (II).
The reaction of resorcinol with a dialkyl trifluoroacetosuccinate in the presence of polyphosphoric acid may suitably be performed at a temperature of about 15-30xc2x0 C.
Typical reaction conditions for the alkylation of a 7-hydroxycoumarin with an alkylhalide are well known to those skilled in the art and include a solvent such as acetone at reflux in the presence of a base such as potassium carbonate.
Typical reaction conditions for ester hydrolysis are well known to those skilled in the art and include dilute hydrochloric acid in methanol or ethanol at reflux
Dialkyl trifluoroacetosuccinates of formula I may be prepared from the corresponding alkyl bromoacetate and alkyl trifloroacetoacetate according to the route described in C. Aubert et al. J. Fluorine Chem. 1989, 44,361. Suitable alkyl bromoacetates and alkyl trifluoroacetoacetates are commercially available.
Since the inhibition of cytochrome P450 enzymes is often the mechanism for drug/drug interactions, the assay according to the invention is particularly useful for identifying compounds which may give rise to adverse drug/drug interactions. The assay can therefore be used in combination with the chemical modification of test compounds to increase a test compound""s potential for use as a pharmaceutical.
Thus according to further aspects of the invention there are provided a method for reducing the CYP2C9 enzyme inhibitory activity of a compound, comprising the steps of identifying the compound as an inhibitor of CYP2C9 in the assay described above; and thereafter producing a chemically modified version of the test compound in which the functionality suspected to be responsible for CYP2C9 inhibition is eliminated or changed; and novel compounds produced according to this method.
The chemical modification of test compounds according to this method can be performed using techniques well known to those skilled in the art.
The novel compounds produced according to this aspect of the invention may find application as pharmaceuticals. A compound produced according to this method will be readily identifiable as novel by performinq routine literature and database searches. The pharmaceutical activity of such compounds can be readily ascertained using conventional biological screening methods known to those skilled in the art.
All publications including, but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
The invention is illustrated by the following examples.